title: Epithelial wounds induce differential phosphorylation changes in response to purinergic and EGF receptor activation.
abstract: Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleotides released after scratch wounding activate purinergic receptors, leading to a distinct phosphorylation profile on epidermal growth factor receptor (EGFR) compared with its natural ligand. ATP induced a 2- to 15-fold phosphorylation increase over control on EGFR Y974, Y1086, and Y1148, with minimal phosphorylation intensity on EGFR Y1173 compared with the level measured in response to epidermal growth factor. Differential phosphorylation induced by epidermal growth factor or ATP was site specific on Src, Shc, phospholipase Cγ, protein kinase C, focal adhesion kinase, paxillin, and mitogen-activated protein kinases 1, 12, and 13. After wounding, the P2Y2 receptor mRNA expression increased, and after knockdown, migration and Ca(2+) mobilization were impaired. To examine phosphorylation mediated by P2Y2, cells were cultured in media containing stable isotope-labeled amino acids, the receptor was knocked down, and the cells were stimulated. Mass spectrometry-based comparison of the phosphorylation profiles of control versus transfected cells revealed a 50-fold decrease in phosphorylation of EGFR Y974 and 1086, with no decrease in Y1173 phosphorylation. A similarfold decrease in Src Y421 and Y446 and paxillin Y118 was detected, indicating the far-reaching importance of the P2Y2 receptor in mediating migration.
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts; Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts.
authors.forename: Amanuel
authors.initials: A
authors.lastname: Kehasse
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts.
authors.forename: Celeste B
authors.initials: CB
authors.lastname: Rich
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts.
authors.forename: Albert
authors.initials: A
authors.lastname: Lee
authors.affiliation: Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts.
authors.forename: Mark E
authors.initials: ME
authors.lastname: McComb
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts; Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts. Electronic address: cecmsms@bu.edu.
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Costello
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts; Department of Ophthalmology, Boston University School of Medicine, Boston, Massachusetts. Electronic address: vickery@bu.edu.
authors.forename: Vickery
authors.initials: V
authors.lastname: Trinkaus-Randall
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling; D002465:Cell Movement; D002478:Cells, Cultured; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051966:Phospholipase C gamma / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D058473:Receptors, Purinergic P2Y2 / Q000235:genetics / Q000378:metabolism*; D014947:Wounds and Injuries / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D058473:Receptors, Purinergic P2Y2; D000255:Adenosine Triphosphate; C512478:EGFR protein, human; D066246:ErbB Receptors; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D019061:src-Family Kinases; D011493:Protein Kinase C; D051966:Phospholipase C gamma